2 GENES ENCODING PROTEINS WITH SIMILARITIES TO RUBREDOXIN AND RUBREDOXIN REDUCTASE ARE REQUIRED FOR CONVERSION OF DODECANE TO LAURIC ACID IN ACINETOBACTER-CALCOACETICUS ADP1

Mutants of Acinetobacter calcoaceticus ADP1 unable to grow on dodecane , but retaining the ability to grow on lauric acid were isolated after ethylmethanesulphonate (EMS) treatment. This growth deficiency was co mplemented by a clone from a gene library constructed from chromosomal DNA of the wild-type strain. The complementing DNA mapped in a gene e ncoding a polypeptide with homology to rubredoxins. The deduced putati ve rubredoxin amino acid sequence is more similar to related proteins from Cram-positive bacteria than to the Pseudomonas oleovorans rubredo xin involved in alkane oxidation. An adjacent gene encodes a protein w ith similarity to rubredoxin reductase from Pseudomonas oleovorans and related NAD(P)-dependent reductases. Disruption of the rubredoxin-enc oding gene by insertion of a Km(R)/lacZ cassette rendered the resultin g strain unable to grow on dodecane or hexadecane. This demonstrates t hat these genes are necessary for alkane degradation. Transcriptional fusion of lacZ to the rubredoxin-encoding gene led to low level consti tutive beta-galactosidase expression, whereas the fusion oriented in t he opposite direction was not expressed.